Ligand-directed labeling of opioid receptors for covalent attachment of fluorophores or small-molecule probes

Summary This protocol describes endogenous labeling of opioid receptors (ORs) using a ligand-directed reagent, naltrexamine-acylimidazole compounds (NAI-X). NAI acts by guiding and permanently tagging a small-molecule reporter (X)—such as fluorophores or biotin—to ORs. Here we detail syntheses and uses of NAI-X for OR visualization and functional studies. The NAI-X compounds overcome long-standing challenges in mapping and tracking endogenous ORs as the labeling can be done in situ with live tissues or cultured cells. For complete details on the use and execution of this protocol, please refer to Arttamangkul et al.1,2


SUMMARY
This protocol describes endogenous labeling of opioid receptors (ORs) using a ligand-directed reagent, naltrexamine-acylimidazole compounds (NAI-X). NAI acts by guiding and permanently tagging a small-molecule reporter (X)-such as fluorophores or biotin-to ORs. Here we detail syntheses and uses of NAI-X for OR visualization and functional studies. The NAI-X compounds overcome long-standing challenges in mapping and tracking endogenous ORs as the labeling can be done in situ with live tissues or cultured cells. For complete details on the use and execution of this protocol, please refer to Arttamangkul et al. 1,2

BEFORE YOU BEGIN
Visualization of functional opioid receptors (ORs) in brain tissues from wild type animals has been challenging due to naturally low receptor expression levels. One frequently used approach is to genetically tag ORs with epitope peptides or fluorescent proteins on the N-or C-terminus of the receptors, and that knock-in mice have been made with these approaches. 3 The development of a chemical labeling approach that resulted in naltrexamine-acylimidazole compounds (NAIs) offers alternative ways to study endogenous ORs from any cells or animals. 1,2 The labeling of ORs with NAI compounds relies on a high affinity antagonist naltrexamine to initiate binding at the orthosteric pocket of receptor, which then guide a chemical bond to take place between the reactive acylimidazole moiety of NAI and a nucleophile of amino acid side chains including Lys, Ser, Thr, Tyr or His on the receptor ( Figure 1A). The reaction yields a permanent attachment of a reporter or fluorescent dye to the receptor. One significant advantage of this approach is the guide-ligand naltrexamine can be simply washed from the receptor binding pocket with any buffers. The targeted nucleophilic amino acids for labeling reside on the extracellular side of receptor and thus the attachment of a dye or reporter at one of these sites is predicted to have minimal perturbation on receptor activation and regulation processes.
Note: One flask will remain un-transfected as a means to determine the duration of antibiotic treatment. 4. Once your flask has reached desired number of cells $11 3 10 6 (35% confluence), transfect cells by preparing a transfecting solution using a $1:9 ratio of OR-expressing plasmid to FLP-recombinase-expressing vector (pOG44). a. In one Eppendorf tube, combine 0.9 mg of the OR-encoding plasmid with Hygromycin B resistance, 7.9 mg pOG44 vector, and 1.75 mL room temperature Opti-MEMä. b. In another Eppendorf tube, combine 17.5 mL lipofectamine reagent and another 1.75 mL of room temperature Opti-MEMä. c. Incubate each mixture at room temperature for 5 min. d. Combine both mixtures and incubate for an additional 20 min at room temperature. 5. Slowly add the transfecting solution dropwise to only one of the T-75 flasks-the one that is designated to be stably transfected.
Note: The other untransfected T-75 flask of cells will be used to determine effectiveness of antibiotic Hygromycin B during selection. 6. 24 h after transfecting, passage cells from each flask to new T-175 flasks with $11 3 10 6 cells ($15% confluence). Note: This step has two goals. First, by passaging half of the cells, the genetic diversity from the initial transfection is maintained and potential issues with clone bias are minimized. Second, cells are seeded at a low density so that they do not become confluent during the antibiotic selection.
7. Add 50 mg/mL Hygromycin B to the media, Gibco High-Glucose DMEM plus 10% FBS, to each flask. 8. Replace media including fresh antibiotic every two days while cells are actively dying.
Note: This process typically lasts 4-6 days. 9. Once cells are no longer actively dying, swap media with antibiotic twice per week. 10. Once the un-transfected flask appears to have no remaining cells and the transfected flask has at least 10 plaques, passage cells for further experimentation, re-seeding, and/or expression validation.
Note: Each plaque is a grouping of clonal cells, all touching, that become visible to the unaided eye upon visual inspection of the flask. The process of FLP-mediated recombination is well known to have low efficiency. In our hands, transfection of a T-75 (and selection in a T-175) results in 10-30 plaques if the construct has minimal to no cellular toxicity. Success of transfection can be measured by recombinant tag systems, like the Flag-tag system used in our recombinant OR expression vectors. We generally label cells with anti-flag M1 antibody directly conjugated with AlexaFluor 647 in the presence of calcium and readout fluorescently labelled receptors by flow cytometry.
Prepare acute brain slices from rodents 11. Anaesthetize a rat or mouse with isoflurane and put down by thoracic percussion or decapitation.
Note: NAI-X labels ORs of any species that bind to naltrexamine. The labeling is expected to be widely useful for any developmental ages or sexes. Examples shown in this protocol use rat or mouse brains from both male and female age 21-28 days.
14. Trim the brain with a razor blade to proper size that contains an area of interest.
Note: We trim the brain such that it will not take too much time during slicing with a vibratome. It is also important that a block of trimmed brain should be large enough to be stably placed on the vibratome plate. The process generally requires trials and practice to achieve healthy brain slices.
15. Use cyanoacrylate glue (e.g., Krazy glue) to fasten the trimmed brain to a vibratome plate. 16. Placed the trimmed brain on vibratome plate in a cutting chamber filled with the oxygenated warm ACSF (30 C-34 C) containing 3 mM (+) MK801. 17. Slice the brain area of interest at 220-or 280-mm thickness using a vibratome (Leica, Nussloch, Germany).

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Note: Thickness of brain slices depends on areas of interest as well as experimental goals. For imaging and electrophysiological purpose, a range of 200-300 mm thickness are suitable for handling and maintain healthiness of brain slices.
Note: The temperature of ACSF during this step can be varied depending on the area of brain. For example, the temperature at 31 C-34 C is suitable to maintain locus coeruleus and midbrain dopamine slices for 6 h whereas the room temperature ($22 C) is good for striatal slices.

Institutional permissions
The use of animals was conducted in accordance with the National Institutes of Health guidelines and the approval of the Institutional Animal Care and Use Committee of OHSU. Animals were separated by sexes and housed as a group (2-3 rats or 2-5 mice) to a cage in the animal care facility. Food and water were available ad libitum on the 12-h light/dark cycle. Dissolve lyophilized powder of 20 nmol NAI-X in dimethylsulfoxide (DMSO) 2 mL. Add 198 mL deionized water.

KEY RESOURCES
Note: NAI-X is generally aliquoted and stored as 20 nmol lyophilized powder in an Eppendorf tube. The combined solvent used to make a solution, 200 mL will result a final concentration of 100 mM.
Note: This 100 mM stock solution can be stored at À20 C for up to 6 months. The working solution should be stored at 4 C and used within $2 weeks.

Artificial cerebrospinal fluid (ACSF) solution
To prepare a liter of ACSF, dissolve the following salts in Milli-Q water. The preparation time of the ACSF is approximately 30 min.
Note: ACSF should be made fresh daily.

Macro zoom fluorescence microscope system
We use an Olympus MVX10 macro zoom fluorescence microscope equipped with a Retiga-2000R camera from QImaging. The magnification range is 0.633 -6.33 with MVPLAPO 13, N.A. 0.25, air objective lens and WHN103 eyepiece. We use Q-Capture software (QCapture 2.98.2) for Image acquisition.

Two-photon microscope
The two-photon microscope is a custom-built apparatus using an upright microscope Olympus BX51W1 and a 603 water immersion Olympus LUMFI, NA 1.1 lens. Excitation of laser generated from Cameleon Ti:sapphire is set at 810-nm for Alexa594 dye. We use Scan Image Software 10 for data acquisition.

Confocal microscope
We use a Nikon Spinning Disk confocal microscope (Laser: 488 nm, Emission filter: 525/36 nm) equipped with a temperature control system and a 1003 oil immersion objective (NA = 1.48, working distance = 0.12 mm).

Image analysis
We use Fiji software 11 for all post hoc analysis of images. All images are set using the same brightness/contrast for clarity.

STEP-BY-STEP METHOD DETAILS
Synthesis of NAI compounds (NAI-X) Timing: 2 days (for step 1) Timing: 3 days (for step 2) Timing: 1 day (for step 3) Timing: 3 days (for step 4) Timing: 2 days (for step 5) There are 5 steps in making NAI compounds. j. Collect the white solid precipitate by filtering the cloudy solution using a fritted glass funnel placed on a filtering flask and apply a house vacuum to filter. k. Wash the solid product 3 times with ice-cold ethanol by adding the solvent to the precipitate in the fritted glass funnel and apply a house vacuum to remove ethanol. l. Place the wet solid product in a 20 mL scintillation vial and dry it by applying a house or high vacuum at room temperature.  Note: To confirm the reaction is completed, the peak of naltrexamine will be absent on the chromatogram.
i. Pour the reaction mixture into a round bottom flask. j. Remove DMF under reduced pressure by applying a vacuum using a Fisher Scientific Maxima C Plus high vac pump connected to a condensing device for liquid collection. k. Dissolve the residue, which appears as a film around the round bottom flask with 5% acetonitrile in water plus 0.1% formic acid for HPLC purification. l. Purify the crude solution with an Agilent 1260 Infinity II semi-preparative HPLC system using a reverse phase Vydac 300Å C8, 10 3 250 mm column, 5 mL/min flow, and a linear gradient over 25 min from 0%-40% acetonitrile in water with 0.1% formic acid. m. Combine pure fractions and lyophilize.
Note: The lyophilized, white powder is obtained at approximately 94% yield of starting material (b-naltrexamine$2HCl) and purity of 97% determined by HPLC.
CRITICAL: The high purity of compound 2 as shown in [Reaction equation 2] is important for the reaction in step iv and v. We routinely check the purity of the lyophilized powder with an Agilent 1260 Infinity II analytical HPLC equipped with a Thermo Scientific Velos LTQ mass spectrometer. Using reverse phase Agilent Pursuit XRs 100Å C18, 4.6 3 250 mm column, 1 mL/min flow and a linear gradient over 15 min from 0%-40% acetonitrile in water with 0.1% formic acid, the product peak will appear at 536.5 (m/z+1) on the electron-spray ionized mass spectrum.  4] should be stored in a moisturecontrolled container at À80 C because it is very sensitive to moisture and will degrade as the result of self-reaction by phenol-OH on naltrexamine moiety. We recommend using compound 4 promptly in the next step.

5.
Conjugation of NAI-AK with Alexa594 azide using copper (I)-catalyzed azide-alkyne cycloaddition (CuAAA) click chemistry. This step is designed to bring in a reporter to the guided ligand naltrexamine. The alkyne functional group in NAI-AK (compound 4) reacts with any azide compounds via copper-catalyzed alkyne azide cycloaddition. 12,13 It is a simple, selective, and high yield reaction that can take place in various solvents e.g., dimethylsulfoxide, dimethylformalmide, acetonitrile or water. These exceptional properties of click reaction allow the conjugation of NAI-AK to a reporter with minimal byproducts. Alexa594 azide (AFdye 594 azide from Click Chemistry Tools) was used here as an example to generate NAI-A594 (see Reaction equation 5 CRITICAL: Since the initial publication, 1 we notice the intermediate naltrexamine-acylimdazole-alkyne (NAI-AK, compound 4) is very unstable in aqueous solution. The click reaction to conjugate fluorescent dyes is therefore done in anhydrous DMSO and tetrakis(acetonitrile)copper(I) tetrafluoroborate is used as a catalyst instead of CuSO 4 and ascorbate.
c. Dissolve compound 4 (2.5 mg, 3.78 mmol) in 450 mL anhydrous DMSO and add to the mixture from b. d. Stir overnight (12-18 h) under argon. e. Check the completion of the reaction by HPLC analysis of the crude material with an Agilent 1260 Infinity II analytical HPLC connected to a Thermo Scientific Velos LTQ mass spectrometer. The analytical HPLC condition uses a reverse phase Agilent Pursuit XRs 100Å C18, 4.6 3 250 mm column and 1 mL/min flow of a linear gradient over 15 min from 5%-100% acetonitrile in water with 0.1% formic acid.
Note: The reaction is completed when Alexa594 azide peak is absent on the chromatogram and the product peak appears at the retention time of 10.6 min.
f. Purify the reaction mixture on an Agilent Infinity preparative HPLC using a reverse phase Agilent Pursuit XRs 100Å C18, 30 3 250 mm column, 45 mL/min flow and a linear gradient over 15 min from 5%-65% acetonitrile in water with 0.1% formic acid.
Note: Any preparative columns can be used for purification with appropriate adjustment of the flow of the solvents. We recommend using a similar packing material to obtain the best separation. Note: The lyophilized, purple powder is obtained at approximately 98% yield with 75% purity determined by HPLC.
h. Check the purity of the lyophilized powder with an Agilent 1260 Infinity II analytical HPLC connected to a Thermo Scientific Velos LTQ mass spectrometer using a reverse phase Agilent Pursuit XRs 100Å C18, 4.6 3 250 mm column, 1 mL/min flow and a linear gradient over 15 min from 5%-60% acetonitrile in water with 0.1% formic acid. A product peak will appear at 755.46 (m/z+2) on the electron-spray ionized mass spectrum.
Note: UV absorption can also be used to monitor the product at 220 and 594 nm. Dual wavelength detection is recommended.
CRITICAL: If only UV detection is available, it is important to check the combined product fractions with mass spectrometry.
CRITICAL: When using other dyes, it may be advantageous to use acetonitrile/water (1:1) with 0.1% trifluoroacetic acid to ensure better solubility after lyophilization. CRITICAL: If absorbance is above 1, make a new dilution until absorbance is below or in the linear range of Beer's Law. This value is important for concentration calculations, which is used to determine the total yield and the amount of material (in mL) when aliquoting into smaller portions for storage.

m. Aliquot in 20 nmol portions and lyophilize.
Note: The scale of this synthesis is based on the commercially availability of 1 mg of the dye. Generally, < 1 mg of product is obtained. Weighing out for making a working stock solution is not practical. The best way to get the proper amount of product for easy use and storage is thus relying on their maximal absorption and the known extinction coefficient. We find that 20 nmol material is sufficient for most applications and that the stock and working solution will be consumed within a practicable time.
CRITICAL: The small aliquot of 20 nmol dried compound should be stored at À20 C with humidity control. See the reconstitution procedure and how to store the working solution in Materials and Methods above.

Validation of NAI-X labeling of opioid receptors in cultured cells
Timing: 2 days (for step 6) Timing: $3 days (For step 7) ll OPEN ACCESS Optimization of the concentration and time used for NAI-X labeling can be guided by labeled cells' fluorescent signal to background ratio (i.e., labeled in the presence of a blocking ligand such as naloxone). For many experimental applications, we have found that a sub-saturating concentration close to EC 50 of NAI-X is adequate to efficiently label the receptor. However, a new assay scheme (i.e., new conjugated fluorophore or cell type) may require re-optimization of NAI-X labeling conditions. Ultimately, decision making for NAI-X labeling protocols can be influenced by OR expression levels, fluorescent dyes, and sensitivity of the detection methods required by the experimental question. For general purposes, NAI-X concentrations can range from 10 nM to 500 nM with incubation timing of 1-4 h depending on concentration of the compound and temperature. Therefore, a concentration response curve is performed to identify optimal labeling conditions. This protocol uses HEK293 cells stably expressing the m-opioid receptors (MOR) as an example.
6. Using flow cytometry to determine specific and irreversible labeling. a. Seed 3 3 10 5 ($20% confluence) cells stably expressing the m-opioid receptors (MOR) (As described in the ''before you begin'' section) into various wells of a 12 well plate.
Note: For every concentration of NAI to be tested, seed an additional well of cells to serve as a background control-which will be labeled with NAI-X in the presence of excess naloxone, 10 mM.
b. Return cells to incubator set to 37 C, 93% humidity, and 5% CO 2 for 48 h to grow to $1 3 10 6 in number (70% confluence). c. Once cells have reached desired confluence, add a final concentration of 10 mM naloxone to all background control wells.
Caution: NAI-X is less stable at basic (> pH 8); ensure media for labeling is pre-equilibrated at pH 7.4.
Caution: Wells should contain equilibrated DMEM+10% FBS; there is no need to exchange for fresh media or remove FBS before labeling.
d. Add a range of concentrations (10-500 nM) of NAI-X to control and test wells. e. Return cells to incubator at 37 C, 93% humidity, and 5% CO 2 . Allow labeling to proceed for 1 h. f. Once labeling is completed, wash each well once with PBS. g. Aspirate PBS and add 500 mL of TrypLE to lift cells. Incubate at room temperature for 4 min or 37 C for 2 min. h. Add 1 mL of Gibcoä High-Glucose DMEM with 10% FBS. i. Gently pipette up and down four times without introducing air bubbles to ensure all cells are lifted from the treated surface of the flask. j. Transfer cells to Eppendorf tubes. k. Spin down cells in Eppendorf tubes at 300 3 g for 2 min. l. Remove supernatant and resuspend cells in 1 mL PBS with 10 mM naloxone.
Note: Addition of naloxone in this step will remove the unreacted NAI-X from orthosteric binding site on the receptor and ensure the fluorescent signal is only from the NAI-X which has undergone covalent labeling.
m. Analyze by flow cytometry, capturing 10,000 events after gating for single cells.
Note: The results from above protocol will be used to construct a concentration response curve and calculate an EC 50 concentration (see Figure 4A). This same protocol can be used to optimize the time of labeling, e.g., 400 nM of NAI-A488 (see Figure 4B) is used at various incubation times. A saturation curve is created to confirm specificity of labeling. The following protocol describes the application of NAI-X in receptor internalization study. a. Plate cells on imaging dishes. We use LabTek II 8 well dishes with borosilicate glass bottoms.
CRITICAL: We have found that even though these LabTek II 8 well dishes are pre-coated, cells better adhere after additional coating with poly-L-lysine, following manufacturer's instructions.
b. Two days later, warm media to 37 C in incubator.
Note: Optional, use imaging media without phenol red, and/or to supplement with HEPES if microscope does not include CO 2 control. The pH of the medium should be around 7.4.
c. Add NAI-A488 to well(s) at 400 nM. d. Return cells to incubator to label for 10 min. e. Wash cells twice with pre-equilibrated media (DMEM without phenol red, supplemented with 20 mM HEPES) and replace with fresh pre-equilibrated media. f. Image on microscope (Nikon Spinning Disk confocal with temperature control at 37 C, 1003 objective, 2-s exposure per image). g. Add agonist and monitor on microscope for internalization. In Figure 5, images taken every 6 min over approximately 20 min.

Validation of NAI-X labeling of opioid receptors in brain slices
Timing: 2-4 h (for step 8) Timing: 1 day (for step 9) NAI-X can fluorescently tag ORs in brain tissues of any animals. The labeling can be done in situ in acutely prepared slices. The labeled slices can be fixed for further study. 8. Labeling of opioid receptors in live brain slices. These steps describe the application of NAI-X in visualizing ORs in acute (freshly prepared) brain slices. a. Prepare brain slices as shown in the ''before you begin'' section.
Note: We made a simple incubation vial (see Figure 3) for economical uses of NAI-X in labeling live brain slices. Most materials are available in the laboratory. The tool allows a small usage of NAI-X while maintaining healthy brain slices during labeling, and thus can be used in subsequent functional assays e.g., electrophysiological recording and receptor internalization.
b. Make an incubating vial using a 10-mL syringe, a small piece of mesh fabric (2 3 2 inches) and a 30-mL scintillation vial ( Figure 3A). i. Cut the tip of 10-mL syringe near the 3 mL mark.
ii. Stretch the net cloth over the opening, secure the cloth with rubber band ( Figure 3B).
iii. Use cyanoacrylate glue (Krazy glue) to fix mesh fabric to barrel of syringe ( Figure 3C). iv. Cut and remove the excess fabric material ( Figure 3D). v. Handle of transfer pipet can be used as a convenient cap ( Figures 3D and 3E). c. Add 4 mL ACSF buffer into the scintillation vial and set up the tool as shown in Figure 3E. d. Insert a small tube for bubbling carbogen gas (5% carbon dioxide + 95% oxygen).
Note: The bubbling tube is outside net chamber, see Figure 3F. e. Add 4 mL of NAI-X to the vial. The concentration of NAI-X is 100 nM. f. Mix well. g. Drop a brain slice inside the net chamber, Figures 3G and 3H.
Caution: More than one slice can be placed in the chamber, but they should not be stacked on top of each other.
Caution: To avoid cell death during this step, be careful not to bubble too close and cause brain slices to swirl.
Note: NAI-A594 is a good choice for two-photon microscopy because Alexa594 dye has a high two-photon absorption at 810 nm.
Note: Similar to cell culture experiments, we recommend using naloxone to confirm specific labeling of ORs. When included in labeling solution of NAI-A594, there will be negligible fluorescent signals on brain slices. Naloxone can also be used to displace residual NAI-A594 in the binding pocket.
Caution: If labeled neurons are subjected for further functional assays, it is important to ensure that naloxone used after labeling is completely washed out with ACSF buffer prior to experiments. Antagonist property of naloxone will hinder the action of test drugs.
i. Remove a labeled slice and submerge it under ACSF in a petri dish. j. Take slices to a macroscope to visualize labeled receptors on large brain areas. Note: A perfusion system provides a convenient way for drug application and washing steps. m. Take images using a 603 water immersion lens (Olympus LUMFI, NA 1.1) and laser at 810 nm excitation.
9. Labeling of opioid receptors for fixed brain slices. These steps describe NAI-X labeling for fixed tissues. a. Prepare brain slices as shown in ''before you begin'' section. b. Incubate brain slices in oxygenated ACSF containing NAI-X at a final concentration of 30 or 100 nM for 1 h at 30 C-34 C. c. Wash the labeled slices, 2 3 10 mL in ACSF. d. Add 4% formaldehyde to labeled slices in Petri Dish.
Note: The volume of 4% formaldehyde can be varied depending on the size of petri dish. Slices should be completely submerged under the fixative solution.
e. Shake slices in a Petri dish using a rotator for 20 min. f. Wash fixed slices in a saline-phosphate buffer twice.

EXPECTED OUTCOMES
NAI-X works to guide and covalently attach reporters like fluorescent dyes or biotin to ORs. The labeling property of NAI-X is concentration-and time-dependent indicating specificity to ORs. It is further confirmed by the blockade of labeling when co-incubating NAI-X in the presence of naloxone,

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a universal opioid antagonist (Figure 4). NAI-X has a relative high binding affinity of NAI-X to ORs hence it may linger in the orthosteric binding pocket of receptor for sometimes, especially after incubating in a saturating concentration of NAI-X. Adding naloxone to samples before flow cytometric measurement will ensure to eliminate the signal of this reversible binding NAI-X as well as verify the covalent tagging of fluorophores (Figure 4). Labeling of opioid receptors with NAI-X can be effectively achieved at EC 50 concentrations of nanomolar ranges. We recommend using this low concentration, which can be easily washed with a buffer for labeling if the labeled receptors will be used later in functional assays.
Labeled ORs by NAI-X are still functional. Agonist-induced receptor endocytosis is a quick and reliable assay to check functionality of the labeled receptors. We observed MOR and DOR internalization in HEK293 cells expressing Flag-MOR and Flag-DOR, respectively using a confocal microscope ( Figures 5A and 5B). With a 2-photon microscope, we observed internalization of endogenous DOR in mouse striatal cholinergic interneurons after application of [Met 5 ]enkephalin (ME) and deltorphin. DAMGO, which is a MOR selective agonist did not induce observable MOR internalization. 2 NAI-X is a useful tool for identifying neurons expressing ORs in live brain tissues. 1 Visualization of ORs can be observed using various microscopes including a macroscope, a confocal microscope and a 2-photon microscope ( Figure 6).
NAI-A594 can efficaciously label MOR and DOR. To differentiate if a neuron expresses each receptor or both, we used selective antagonist of each receptor e.g., CTAP for MOR and SDM25N for DOR to block labeling of each receptor subtype. Figure 7 shows examples of labeling OR subtypes on a single neuron co-expressing MOR and DOR of cholinergic interneurons from ChAT(BAC)-eGFP transgenic mice. 2 The cholinergic interneurons from these mice expressed green fluorescent protein via the regulation of choline acetyltransferase gene (shown in Figure 7 as blue cells). Co-incubation of NAI-A594 with a selective MOR antagonist CTAP was used to display labeling of DOR on a cholinergic interneuron of the striatum. Alternatively, labeling MOR with NAI-A594 was done in the presence of a selective DOR antagonist SMD25N (Figure 7).
NAI-labeled ORs in brain slices can be fixable. This application is useful for anatomical studies. Figure 8 shows examples labeled ORs of fixed striatal slices. As labeling of this reagent relies on proper conformations of receptors, it is essential to label live brain tissues before fixation (see step 9).

LIMITATIONS
Although NAI-X is a small molecule and can penetrate deeper in the tissues than antibodies, fluorescent signals significantly decrease after 100 mm from the top surface. The optimal working range for best signals is found to be between 10-50 mm from the surface.

Problem 1
Labeling receptors with NAI-X could potentially perturb receptor functions. This is a common problem for any modified receptors including genetically or chemically tagged receptors that should be investigated. The linker in the NAI-X molecule was designed with the goal of allowing labeling of the receptor on its extracellular loops, thus minimizing the potential for the covalently bound fluorophore to disrupt receptor function that occurs via interaction with signaling proteins in the cytoplasm. We found the labeled receptors were functional as shown by activation of G protein-gated inwardly rectifying potassium channels of labeled MOR in a locus coeruleus neurons 1 , DOR internalization in cholinergic interneurons of the striatum 2 , and ME-induced MOR and DADLE-induced DOR internalization in HEK293 cells (Figures 5A and 5B). However, it remains possible that a fluorescent dye on the receptor may perturb the accessibility and interaction of agonists at the binding pocket. It is therefore important to systematically examine the functions of labeled receptors and compare the results with unlabeled receptors in each system of interest.

Potential solution
Construct and compare EC 50 's and maximal responses of labeled and unlabeled receptors in the assay of interest. The assays should include but not limit to recruitment of mini-G proteins and/or arrestins, activation of G protein-gated inwardly rectifying potassium channels, and production of cAMP. We recommend using a set of well-known full agonists and partial agonists to validate receptor functions.

Problem 2
Trafficking of labeled receptors in the absence of agonist activation (see step 7).
A potential issue that could occur in visualization of NAI-X labeled ORs in cell culture is intracellular fluorescence signals before agonist activation of ORs. Importantly, NAI-A594 or NAI-A488 is not expected to permeabilize and label intracellular receptors. When long incubation of NAI compounds is done at 37 C, fluorescent puncta are observed without agonist activation. The problem is likely due to the labeled ORs undergoing constitutive endocytosis during incubation time.

Potential solution
Incubate cells with a high concentration of NAI-X for a shorter time e.g., 10 min. A near saturating concentration is recommended for this fast labeling. This is suitable for cell cultures where receptors on a monolayer of cells can quickly react with NAI-X. Additionally, the labeling with this high concentration of NAI-X can also be done at room temperature or on ice to prevent endocytosis. It is also important to keep in mind that NAI-X is a reversible antagonist and thus it is important to thoroughly wash this reagent from samples prior to functional assays. When brain slices are used, incubating slices near EC 50 concentration of NAI-X is necessary. Brain slices contain various cell types, and a high concentration of NAI-X may cause substantial non-specific labeling. Incubating slices with NAI-X at room temperature can minimize constitutive endocytosis.
The labeling of NAI-X is dependent on proper conformations of the receptor for the ligand to bind and trigger the reaction. Labeling fixed cells or tissues with NAI-X will potentially yield a lower quality of image compared to labeling live tissues.

Potential solution
Label freshly prepared brain slices before fixation process.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Seksiri Arttamangkul (arttaman@ohsu.edu) and Braden Lobingier (lobingib@ohsu.edu).
Materials availability NAI-A594 and NAI-488 generated in this study can be made available on request to the Medicinal Chemistry Core Facility at Oregon Health and Science University. There will be fees for service depending on the amount of the material. Additionally, chemists at the core facility can customize the reagents for different reporters or fluorescent dyes. Please contact the core director, Dr. Aaron Nilsen (nilsena@ohsu.edu).